The human serum proteins albumin (ALB), .alpha.-fete-protein (AFP) and vitamin D binding protein (VDB) are known to be members of a multigene ALB family. All three proteins are found in serum where they mediate the transport of a wide variety of ligands. ALB binds fatty acids, amine acids, steroids, glutathione, metals, bilirubin, lysolecithin, hematin, prostaglandins and pharmaceuticals (for review, see 1). AFP binds fatty acids, bilirubin and metals (2, 3). VDB binds vitamin D and its metabolites as well as fatty acids, actin, C5a and C5a des Arg (4-7).
In addition to their transport capabilities, ALB family proteins possess a wide assortment of other functional activities. ALB is the main contributor to the colloid oncotic pressure of plasma, acts as a scavenger of oxygen-free radicals and can inhibit copper-stimulated lipid peroxidation, hydrogen peroxide release, and neutrophil spreading (1, 8-10). AFP has been implicated in the regulation of immune processes (11-14) and VDB can act as a co-chemotactic factor for neutrophils (6, 15) and as an activating factor for macrophages (16).
The serum levels of ALB family proteins are also known to be responsive to various pathological conditions. ALB is a negative acute phase protein (17) whose levels decrease in times of stress. AFP levels are elevated in women carrying fetuses with certain developmental disorders (18, 19) and in individuals with hepatocarcinoma, teratocarcinoma, hereditary tyrosinemia or ataxia-telangiectasia (20-24). VDB levels are decreased in patients with septic shock (25) or fulminant hepatic necrosis (26, 27).
ALB family members also have significant structural similarities. Homology has been observed at the primary amino acid sequence level and there is also a well-conserved pattern of Cys residues which predicts similar secondary structures (28-32). ALB family genes have similar exon/intron organizations (33-36) and all have been mapped to human chromosome 4 within the region 4q11-q22 (37, 38).
Human "Afamin" (abbreviated as "AFM") is a novel serum protein with a molecular weight of 87000 daltons. It shares strong similarity to albumin family members and has the characteristic pattern of disulfide bonds observed in this family. In addition, the gene maps to chromosome 4 as do other members of the albumin gene family. Thus, AFM is the fourth member of the albumin family of proteins. AFM cDNA was stably transfected into Chinese hamster ovary cells and recombinant protein (rAFM) was purified from conditioned medium. Both rAFM and AFM purified from human serum react with a polyclonal antibody that was raised against a synthetic peptide derived from the deduced amino acid sequence of AFM. It is expected that AFM will have properties and biological activities in common with ALB, AFP, and VDB.
Publications relating to "Background of the Invention" PA0 1. Peters, T. Jr., Adv. Protein Chem. 37, 161-245 (1985). PA0 2. Parmelee, D. C., Evenson, M. A., and Deutsch, H. F. J. Biol. Chem. 253, 2114-2119 (1978). PA0 3. Berde, C. B., Nagai, M., Deutsch, H. F., J. Biol. Chem. 254, 12609-12614 (1979). PA0 4. Daiger, S. P., Schanfield, M. S., and Cavalli-Sforza, L. L., Proc. Nat. Acad. Sci. U.S.A. 72, 2076-2080 (1975). PA0 5. Van Baelen, H., Bouillon, R., and De Moor, P., J. Biol. Chem. 255, 2270-2272 (1980). PA0 6. Kew, R. R., and Webster, R. O., J. Clin. Invest. 82, 364-369 (1988). PA0 7. Williams, M. H., Van Alstyne, E. L., and Galbraith, R. M., Biochem. Bipophys. Res. Commun. 153, 1019-1024 (1988). PA0 8. Holt, M. E., Ryall, M. E. T., and Campbell, A. K., Br. J. exp. Path. 65, 231-241 (1948). PA0 9. Gutteridge, J. M. C., Biochim. Biophys ACTA 869, 119-127 (1986). PA0 10. Nathan, C., Xie, Q-W., Halbwachs-Mecarelli, L. and Jin, W. W., J. Cell Biol. 122, 243-256 (1993). PA0 11. Yachnin, S., Proc. Natl. Acad. Sci. U.S.A. 73, 2857-2861 (1976). PA0 12. Auer, I. O., and Kress, H. G., Cell. Immunol. 30, 173-179 (1977). PA0 13. Alpert, E., Dienstag, J. L., Sepersky, S., Littman, B., and Rocklin, R., Immunol.Commun. 7, 163-185 (1978). PA0 14. Chakraborty, M., and Mandal, C., Immunol. Invest. 22, 329-339 (1993). PA0 15. Perez, H. D., Kelly, E., Chenoweth, D., and Elfman, F., J. Clin. Invest. 82, 360-363 (1988). PA0 16. Yamamoto, N. and Homma, S., Proc. Natl. Acad. Sci. U.S.A. 88, 8539-8543 (1991). PA0 17. Koj, A., in The Acute Phase Response to Injury and Infection 1 (Gordon, A. H., and Koj, A. eds) pp. 45-160 (1985). PA0 18. Brock, D. J. H., and Sutcliffe, R. G., Lancet 2, 197-199 (1972). PA0 19. Allan, L. D., Ferguson-Smith, M. A., Donald, I., Sweet, E. M. and Gibson, A. A. M., Lancet 2 522-525 (1973). PA0 20. Waldmann, T. A., and McIntire, K. R., Lancet 2, 1112-1115 (1972). PA0 21. Belanger, L., Belanger, M., Prive, L., Larochelle, J., Tremblay, M., and Aubin, G., Pathol. Biol. 21, 449-455 (1973). PA0 22. Belanger, L., Pathol. Biol. 21, 457-463 (1973). PA0 23. Ruoslahti, E., Pihto, H., and Seppala, M., Transplant. Rev. 20, 38-60 (1974). PA0 24. Tamaoki, T., and Fausto, in Recombinant DNA and Cell Proliferation (Stein G. and Stein J. eds.) pp. 145-168 (1984). PA0 25. Lee, W. M., Reines, D., Watt, G. H., Cook, J. A., Wise, W. C., Halushka, P. V., and Galbraith, R. M., Circ. Shock. 28, 249-255 (1989). PA0 26. Young, W. O., Goldschmidt-Clermont, P. J., Emerson, D. L., Lee, W. M., Jollow, D. J., and Galbraith, R. M., Lab. Clin. Med. 110, 83-90 (1987). PA0 27. Goldschmidt-Clermont, P. J., Lee, W. M., and Galbraith, R. M., Gastroenterology 94, 1454-1458 (1988). PA0 28. Lawn, R. M., Adelman, J., Bock, S. C., Franke, A. E., Houck, C. M., Najarian, R. C., Seeburg, P. H., and Wion, K. L., Nucleic Acid Res. 9, 6103-6114 (1981). PA0 29. Dugaiczyk, A., Law, S. W., and Dennison, O. E., Proc. Natl. Acad. Sci. U.S.A. 79, 71-75 (1982). PA0 30. Morinaga, T., Sakai, M., Wegmann, T. G., and Tamaoki, T., Proc. Natl. Acad. Sci. U.S.A. 80, 4604-4608 (1983). PA0 31. Cooke, N. E. and David, E. V., J. Clin. Invest. 76, 2420-2424 (1985). PA0 32. Yang, F., Brune, J. L., Naylor, S. L., Cupples, R. L., Naberhaus, K. H., and Bowman, B. H., Proc. Natl. Acad. Sci. U.S.A. 82, 7994-7998 (1985). PA0 33. Sakai, M., Morinaga, T., Urano, Y., Watanabe, K., Wegmann, T. G., and Tamaoki, T., J. Biol. Chem. 260, 5055-5060 (1985). PA0 34. Minghetti, P. P., Ruffner, D. E., Kuang, W-J., Dennison, O. E., Hawkins, J. W., Beattie, W. G., and Dugiaczyk, A., J. Biol. Chem. 261, 6747-6757 (1986). PA0 35. Gibbs, P. E. M., Zielinski, R., Boyd, C., and Dugaiczyk, A., Biochemistry 26, 1332-1343 (1987). PA0 36. Witke, W. F., Gibbs, P. E. M., Zielinski, R., Yang, F., Bowman, B. H., and Dugaiczyk, A., Genomics 16, 751-754 (1993). PA0 37. Mikkelsen, M., Jacobsen, P. and Henningsen, K., Hum. Hered. 27, 105-107 (1977). PA0 38. Harper, M. E., and Dugaiczyk, A., Am. J. Hum. Genet. 35, 565-572 (1983) . PA0 38. Peters, Theodore in ALBUMIN An Overview and Bibliography, Second Edition, 1992. PA0 39. American Hospital Formulary Service Drug Information, Blood Derivatives, 762-763 (1992). PA0 40. Yamashita, T., et al., Biochem. Biophys. Res. Commun. 191 (2), 715-720 (1993). PA0 41. Candlish, John K., Pathology 25, 148-151 (1993). PA0 42. Ohkawa, K., et al., Cancer Research 54, 4238-4242 (1993). PA0 43. He, Xiao Min and Carter, Daniel C., Nature 358, 209-215 (1992). PA0 44. Brown, J. M., et al., Inflammation 13 (5), 583-589 (1989). PA0 45. Emerson, T. E., Critical Care Medicine 17 (7), 690-694 (1989). PA0 46. Halliwell, Barry, Biochem. Pharmacol. 37 (4), 569-571 (1988). PA0 47. Holt, M. E., et al., Br. J. Exp. Path. 65, 231-241 (1984). PA0 II. Alpha Fetoprotein PA0 48. Suzuki, Y., et al., J. Clin. Invest. 90, 1530-1536 (1992). PA0 49. Sakai, M., et al., J. Biol. Chem. 260 (8), 5055-5060 (1985). PA0 III. Vitamin-D Binding Protein PA0 51. Watt, G. H., et al., Circulatory Shock 28, 279-291 (1989).
The sections below contain a summary of background information that is currently available on ALB, AFP, and VDB and contains lists of additional publications relating to these known proteins.
I. Human Serum Albumin
Human serum albumin is an important factor in the regulation of plasma volume and tissue fluid balance through its contribution to the colloid osmotic pressure of plasma. Albumin normally constitutes 50-60% of plasma proteins and because of its relatively low molecular weight (66,300-69,000), exerts 80-85% of the colloidal osmotic pressure of the blood.
The best known functions of ALB involve regulation of transvascular fluid flux and hence, intra and extravascular fluid volumes and transport of lipid and lipid-soluble substances. ALB solutions are frequently used for plasma volume expansion and maintenance of cardiac output in the treatment of certain types of shock or impending shock including those resulting from burns, surgery, hemorrhage, or other trauma or conditions in which a circulatory volume deficit is present. Transfusions of whole blood or red blood cells also may be necessary, depending on the severity of red blood cell loss.
Intravenous (IV) administration of concentrated ALB solutions causes a shift of fluid from the interstitial spaces into the circulation and a slight increase in the concentration of plasma proteins. When administered IV to a well-hydrated patient, each volume of 25% ALB solution draws about 3.5 volumes of additional fluid into the circulation within 15 minutes, reducing hemoconcentration and blood viscosity. In patients with reduced circulating blood volumes (as from hemorrhage or loss of fluid through exudates or into extravascular spaces), hemodilution persists for many hours, but in patients with normal blood volume, excess fluid and protein are lost from the circulation within a few hours. In dehydrated patients, ALB generally produces little or no clinical improvement unless additional fluids are administered.
Although ALB contains some bound amino acids, it provides only modest nutritive effect. ALB binds and functions as a carrier of intermediate metabolites (including bilirubin), trace metals, some drugs, dyes, fatty acids, hormones, and enzymes, thus affecting the transport, inactivation, and/or exchange of tissue products.
ALB is also involved in a number of other vital functions, some of which have only recently been suggested and perhaps others which are as yet unrecognized. Among recognized unique features of albumin are: a) binding, and hence, inactivation of toxic products; b) regulation of the plasma and interstitial fluid concentrations of endogenous and exogenously administered substances and drugs; c) involvement in anticoagulation; d) maintenance of microvascular permeability to protein; and e) scavenging of free radicals and prevention of lipid peroxidation. This latter property may prove to be critically important, particularly in inflammatory disease states in which free radicals are thought to be a major culprit in direct damage due to tissue oxidation, and indirect tissue damage due to inactivation of important antiproteinases such as a.sub.1 -PI and AT-III.
The following is a more detailed summary of the many uses for ALB that have been reported in the literature:
A. Functions of ALB
Contributes to colloid osmotic pressure and thus prevents water loss from circulation;
Aids in transport, distribution, metabolism of fatty acids (primarily long chain), amino acids (Cys and Trp), steroids, glutathione, metals (Ca, Zn), bilirubin, lysolecithin, hematin, prostaglandins and pharmaceuticals to liver, intestine, kidney and brain presumably through specific albumin receptors that have been identified on the endothelium;
Serves as a reservoir for fatty acids intra and extravascularly (60% of the ALB is found extravascularly);
Modification of doxorubicin (DXR) by conjugating it to bovine serum albumin (BSA) improved chemotherapeutic efficiency of DXR presumably by decreasing efflux of BSA-DXR compared to DXR alone (in animal models), suggesting a similar use with ALB;
Inhibits Cu-stimulated lipid peroxidation and hemolysis of erythrocyte membranes (acts as antioxidant);
Scavenges HOCl and peroxy radicals;
Prevents peroxidation of fatty acids by binding to them;
May exert a protective effect in body fluids that have little endogenous antioxidant protection (e.g., eye and cerobrospinal fluids);
In urine, high levels of ALB are diagnostic for detection of early renal pathology in diabetics;
Administered to combat shock and given to neonates with respiratory distress syndrome;
Administered as a vehicle for hematin to treat acute intermittent porphyria;
Used in tissue culture in place of whole serum;
Enhances effectiveness of superoxide dismutase (SOD) when coupled to SOD through enhanced serum half-life;
In microsphere form, ALB is useful as a carrier of therapeutic agents;
Inhibits hydrogen peroxide release and neutrophil spreading.
B. publications relating to ALB
Alpha-fetoprotein (AFP; molecular weight 70,000) is a major serum protein produced during development and is produced primarily by the fetal liver and yolk sac cells. Its synthesis decreases markedly after birth and only trace amounts are present in the serum of adults. Increased adult serum levels are a sign of hepatoma or yolk sac tumor, since these tumors produce AFP. The specific associations of AFP with fetal development as well as the above type of malignancies has attracted much interest and many studies have been done on the structure of AFP and its gene, the regulation of gene expression, and biological functions.
Similar to ALB, AFP has been shown to bind various ligands such as unsaturated fatty acids, estrogens, bilirubin, copper and nickel ions, and others. AFP also has been claimed to regulate immune processes in a variety of systems from many different laboratories, although the results are controversial.
The following is a more detailed list of uses for AFP that are available in the literature:
A. Functions of AFP
Binds unsaturated fatty acids, estrogens, bilirubin, Cu, Ni;
Elevated levels in amniotic fluid of pregnant women indicative of fetal malformations;
High levels also found in hereditary tyrosinemia and ataxia-telangiectasia (autosomal recessive disorder characterized by a defect in tissue differentiation of thymus and liver);
Inhibits NK cell activity;
Induces T suppressor cells;
Inhibits mitogenic responses of lymphocytes to PHA and ConA;
Inhibits T cell proliferation to Ia determinants;
Decreases macrophage phagocytosis and Ia expression;
Inhibits FSH-mediated estradiol production by porcine granulosa cells;
Enhances growth-factor mediated cell proliferation of porcine granulosa cells.
B. Publications relating to AFP
50. EPO Patent Application No. 0353814, Feb. 7, 1990.
The group-specific component (Gc; VDB) is an a.sub.2 -globulin of molecular weight 51,000. It is synthesized in the liver and is the major vitamin D-binding protein in plasma. VDB appears in human populations as three common genetic phenotypes: Gc1, Gc2, and Gc2-1. VDB has also been reported to bind G-actin and to be spatially associated with IgG on lymphocyte membranes.
The following is a more detailed list of uses for VDB that are available in the literature:
A. Functions of VDB
Binds seco-steroid, vitamin D and the derivatives 25-hydroxy vitamin D and 1,25 hydroxy vitamin D, possibly for transport in plasma;
1,25 vitamin D can differentiate monocytes and VDB prevents this;
Binds actin (prevents assembly of actin polymers);
Binds unsaturated fatty acids (e.g., arachidonic acid);
Binds C5a and C5a des Arg to act as a cochemotactic factor for neutrophils;
Acts as an activating factor for macrophages.
B. Publication relating to VDB
The protein of the present invention, AFM, bears a strong similarity in structure to ALB, AFP, and VDB, and is therefore expected to share the above utilities and activities with the known proteins discussed above.